Acidic organelles are present in all cells and tissues of mammalian, plant, yeast and fungal cells, except red blood cells. Many bacteria also contain acidic compartments. These acidic organelles are often involved in metabolism and catabolism of foreign molecules that are brought into the cell by endocytosis. They are often the first line of defense against foreign bacterial or viral infection. The acidic pH of endosomes is critical to the process by which lipid-enveloped viruses enter the cytoplasm after their cellular uptake by receptor-mediated endocytosis. Phagocytosis is the process where extra cellular particles such as bacteria, are engulfed in the cell and then fused to lysosomes for digestion. Acidic organelles have also been shown to be responsible for digestion of high molecular weight proteins, oligosaccharides, glycolipids or peptides by the cell. In addition, they are often involved in therapeutic drug metabolism. Among the cellular organelles that have been found to mediate their enzyme activities by acidification are lysosomes, acidic endosomes, phagosomes, clathrin-coated vesicles and Golgi vescicles.
The Golgi Apparatus and the Endoplasmic Reticulum (ER) are dynamic organelles involved in the synthesis, processing and sorting of cellular proteins and lipids. The Golgi and ER are also involved in a great number of cellular processes including proprotein activation, glycoconjugate modification, glycolipid synthesis, phosphorylation and sulfation of proteins and sugars as well as sorting and trafficking of newly synthesized proteins internally or as part of the secretory pathway. Both organelles have been implicated in defense against pathogens, cholesterol homeostasis, metabolism, apoptosis and cell signaling. As such, the Golgi and the ER contain a great number of metabolic and regulatory enzymes that are involved in ordered function and processing of nearly all proteins, lipids, and carbohydrates in the cell. And more information is continually accumulating that functional deficiencies or mutations in these enzymes are indicative of a number of disease states, particularly those that affect the nervous system such as Alzheimer's Disease, Parkinson's Disease, ALS, Lowe Syndrome, I Cell Disease, Globoid Cell Leukodystrophy, Adrenoleukodystrophy, Niemann-Pick Disease, Metachromatic Leukodystrophy, Mannosidosis and Cystic Fibrosis as well as proliferative diseases, viral infections and neoplastic growth and metastasis.
Prior stains, methods and assays for visualizing acidic organelles are not useful for monitoring enzyme activities in living cells. For example, weakly basic amines have been shown to selectively accumulate in cellular compartments with low internal pH. When further linked to chromogenic or fluorogenic probes, they can be used to label these compartments. Among these is the frequently used acidotropic probe, N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, dihydrochloride (hereafter referred to as DAMP). The fluorescent dyes neutral red and acridine orange are also commonly used for staining acidic organelles, but they lack specificity and are not well retained in the organelles, particularly after fixing and permeabilization.
In particular, methods to analyze Golgi and ER enzyme functional activity within living cells have been hampered by the inability to monitor such activity inside individual organelles. The instant invention intends to address this problem by the use of new, organelle-targeted, live-cell assay systems for Golgi and ER enzyme activities that can be used for discovery and testing of new therapeutic agents for neurodegenerative, proliferative, cardiac diseases or infections. Many ER and Golgi enzymes are under investigation as drug targets for affecting intracellular processing and synthesis mechanisms and thereby provide novel therapeutics for the mentioned and other related diseases.
Tools for staining the Golgi or ER using fluorescence microscopy include recombinant fluorescent proteins that have luminal targeting peptide sequences for the Golgi and ER as well as dye derivatives (e.g. Brefeldin A (BFA) conjugates) that are selectively localized on the cytosolic face of the Golgi and ER in many different cell lines. The peptide sequences identified to target recombinant proteins have also been used to target fluorophores to the Golgi and ER in live cell assays. These dyes are retained in the Golgi and ER due to the presence of specific C-terminal signal sequences such as KDEL (SEQ ID NO:1) or SDYQRL (SEQ ID NO:2) or related sequences that are inserted into the luminal face of the ER or trans-Golgi network. Fluorescently labeled peptides containing these sequences can visualize intracellular processes and molecular interactions at a single-cell level. Protein systems containing these targeting sequences have been used to measure the intra-organelle pH of the ER and Golgi (Wu, et al., 2000). Many other peptide sequences have also been identified for targeting to other organelle structures within living cells. The present invention proposes to utilize these established systems to target fluorogenic enzyme substrates to specific organelles for live-cell analysis of enzyme activity.
The present invention utilizes fluorogenic enzyme substrates for the labeling and tracing metabolic and defective activities in these organelles in live cells or in cell-free systems. These new substrates selectively accumulate in cellular compartments based upon selective targeting sequences and can be used to investigate the enzyme levels responsible for biosynthesis, degradation and recycling of cellular components and for measuring specific enzyme defects involved in a number of human diseases linked to enzyme activity in particular organelles within live cells.
Peptide motifs that can be used to target proteins or even small molecules to various locations within cells are known in the art. For example, the nuclear targeting sequence from the SV40 large T antigen PKKKRKV SEQ ID NO:3 has been used to localize exogenously delivered macromolecular conjugates to live cells as well as recombinant proteins expressed after plasmid or viral DNA transfection/transduction.
Peptide localization motifs have also been described for organelles other than the nucleus. For example, the four amino acid sequence KDEL (SEQ ID NO:1) at the amino terminus of a protein is a well-established ER-retention sequence while the carboxy-terminal sequence of amino acids containing SKL has been identified for peroxisomal targeting. These and other targeting sequences have been used for fluorescent labeling of specific organelles in live cells as an orthogonal method to cell staining by conjugation to small molecule organic dyes. These peptide sequences are known to be actively transported into living cells by the method of retrograde transport
Recently these same peptide motifs have been cloned into fluorescent proteins for specific staining of individual organelles when expressed inside living cells. While such targeting peptides or proteins have been used as research tools for staining specific organelles their potential for real time analysis of enzyme activity within living cells or tissues remains unexplored.
The organelle targeted enzyme substrates of this invention are designed to provide high fluorescence at lower pH values found in some organelles and contain further derivatized groups to support membrane permeation through both outer and organelle membranes of intact cells. They can be used for monitoring enzyme activity in cells at very low concentrations and are not toxic to living cells or tissues. The instant dyes, substrates and methods for their use are described herein for investigating metabolism, monitoring enzyme activities associated with diseases, for analysis of the biogenesis or degenerative states of organelles, the development of, investigating development of cells or cultured neurons, and detecting pH gradients. The current invention is also useful for labeling non-mammalian cells that possess the targeted organelles, including bacteria, yeast, spermatozoa and plant cells, or for the labeling and analysis of enzyme activity in symbiotic or invasive bacterial species that predominantly partitions to specific organelles within living cells.